ABSTRACT The yeast GCN5 gene encodes the catalytic subunit of a nuclear histone acetyltransferase and is part of a high-molecular-weight complex involved in transcriptional regulation. In this paper we show that full activation of the HO promoter in vivo requires the Gcn5 protein and that defects in this protein can be suppressed by deletion of the RPD3 gene, which encodes a histone deacetylase. These results suggest an interplay between acetylation and deacetylation of histones in the regulation of the HO gene. We also show that mutations in either the H4 or the H3 histone gene, as well as mutations in the SIN1 gene, which encodes an HMG1-like protein, strongly suppress the defects produced by the gcn5 mutant. These results suggest a hierarchy of action in the process of chromatin remodeling.

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Elp3 and Gcn5 are histone acetyltransferases (HATs) that function in transcription as subunits of Elongator and SAGA/ADA, respectively. Here we show that mutations that impair the in vitro HAT activity of Elp3 confer typical elp phenotypes such as temperature sensitivity. Combining an elp3 mutation with histone H3 or H4 tail mutations confers lethality or sickness, supporting a role for Elongator in chromatin remodelling in vivo. gcn5elp3 double mutants display a number of severe phenotypes, and similar phenotypes result from combining the elp mutation with mutation in a gene encoding a SAGA-specific, but not an ADA-specific subunit, indicating that Elongator functionally overlaps with SAGA. Because concomitant active site alterations in Elp3 and Gcn5 are sufficient to confer severe phenotypes, the redundancy must be specifically related to the HAT activity of these complexes. In support of this conclusion, gcn5elp3 phenotypes are suppressed by concomitant mutation of the HDA1 and HOS2 histone deacetylases. Our results demonstrate functional redundancy among transcription-associated HAT and deacetylase activities, and indicate the importance of a fine-tuned acetylation–deacetylation balance during transcription in vivo.

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Complex genetic and physiological variations as well as environmental factors that drive emergence of chromosomal instability, development of unscheduled cell death, skewed differentiation, and altered metabolism are central to the pathogenesis of human diseases and disorders. Understanding the molecular bases for these processes is important for the development of new diagnostic biomarkers, and for identifying new therapeutic targets. In 1973, a group of non-histone nuclear proteins with high electrophoretic mobility was discovered and termed High-Mobility Group (HMG) proteins. The HMG proteins include three superfamilies termed HMGB, HMGN, and HMGA. High-mobility group box 1 (HMGB1), the most abundant and well-studied HMG protein, senses and coordinates the cellular stress response and plays a critical role not only inside of the cell as a DNA chaperone, chromosome guardian, autophagy sustainer, and protector from apoptotic cell death, but also outside the cell as the prototypic damage associated molecular pattern molecule (DAMP). This DAMP, in conjunction with other factors, thus has cytokine, chemokine, and growth factor activity, orchestrating the inflammatory and immune response. All of these characteristics make HMGB1 a critical molecular target in multiple human diseases including infectious diseases, ischemia, immune disorders, neurodegenerative diseases, metabolic disorders, and cancer. Indeed, a number of emergent strategies have been used to inhibit HMGB1 expression, release, and activity in vitro and in vivo. These include antibodies, peptide inhbitiors, RNAi, anti-coagulants, endogenous hormones, various chemical compounds, HMGB1-receptor and signaling pathway inhibition, artificial DNAs, physical strategies including vagus nerve stimulation and other surgical approaches. Future work further investigating the details of HMGB1 localizationtion, structure, post-translational modification, and identifccation of additional partners will undoubtedly uncover additional secrets regarding HMGB1's multiple functions.

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Three families of prolyl isomerases have been identified: cyclophilins, FK506-binding proteins (FKBPs) and parvulins. All 12 cyclophilins and FKBPs are dispensable for growth in yeast, whereas the one parvulin homolog, Ess1, is essential. We report here that cyclophilin A becomes essential when Ess1 function is compromised. We also show that overexpression of cyclophilin A suppresses ess1 conditional and null mutations, and that cyclophilin A enzymatic activity is required for suppression. These results indicate that cyclophilin A and Ess1 function in parallel pathways and act on common targets by a mechanism that requires prolyl isomerization. Using genetic and biochemical approaches, we found that one of these targets is the Sin3–Rpd3 histone deacetylase complex, and that cyclophilin A increases and Ess1 decreases disruption of gene silencing by this complex. We show that conditions that favor acetylation over deacetylation suppress ess1 mutations. Our findings support a model in which Ess1 and cyclophilin A modulate the activity of the Sin3–Rpd3 complex, and excess histone deacetylation causes mitotic arrest in ess1 mutants.

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Genetics Inc.), with the exception of the HO probe, which was obtained as a2.6-kb HindIII fragment from the plasmid pGAL-HO (9).Other methods. Yeast cells were transformed by the LiOAc method (6).?-Galactosidase assays were performed as described elsewhere (26).RESULTSThe Gcn5 protein is required for HO expression. HO geneexpression is dependent on SWI5. This gene encodes a zincfinger DNA-binding protein which binds specifically, alongwith the PHO2 protein, to the upstream region of the HOpromoter (1, 34). Genetic studies have described a series ofextragenic suppressor mutations that permit expression of HOin the absence of the SWI5 gene product (17, 33). Two of thegenes identified in this screen, RPD3 and SIN3, encode, re-spectively, a histone deacetylase and a protein tightly associ-ated with it (10, 29, 35). The fact that mutations in the genepair SIN3/RPD3 are able to suppress the absence of the Swi5protein suggests that one of the roles of the Swi5-Pho2 het-erodimer is the recruitment, either directly or indirectly, of ahistone acetyltransferase activity. A likely candidate is theGCN5 gene, which encodes a protein with histone acetyltrans-ferase activity (13). To test this idea, we examined the levels ofHO mRNA produced in wild-type and isogenic gcn5 mutantstrains (obtained by disruption of the GCN5 gene; see Mate-rials and Methods). We found that a gcn5 mutant strain pro-duced significantly less HO mRNA (Fig. 1A). By contrast, theabsence of the Gcn5 protein did not impair the normal levelsof PHO2 and SWI5 mRNA.In principle, SWI5 and GCN5 gene products could act in thesame pathway or through different pathways to activate HOexpression. If two genes act in the same pathway, then thephenotype of the double mutant should be the same as that ofone of the single mutants. On the other hand, if two genes actthrough different pathways, then the phenotype of the doublemutant should be more severe than that of either single mu-tant. To distinguish between these two possibilities, we mea-sured the ?-galactosidase activity produced by a chromosomalHO-lacZ gene fusion in a swi5 gcn5 double mutant and com-pared it to those in the single mutants (Fig. 1B). HO-lacZexpression in the gcn5 and swi5 mutants was reduced 50- and200-fold, respectively. In the double mutant, HO-lacZ expres-sion was reduced 200-fold. The ?-galactosidase values of theswi5 mutant are so low (0.5 Miller units) that we cannot makea conclusive argument about the relationship of SWI5 andGCN5. However, since both defects are suppressed by thesame mutations (i.e., by rpd3, sin1, and sin2 mutations; seebelow) and since the levels of mRNA for SWI5 and PHO2genes are not affected by gcn5 mutations (Fig. 1A), these factssupport the idea that SWI5 and GCN5 act in the same pathwayto stimulate HO expression.Deletion of the RPD3 gene suppresses the gcn5 mutation.The results described above are compatible with the idea thathistone acetylation is required for maximal HO transcriptionalactivation. According to this hypothesis, a mutation in a geneencoding a deacetylase should be able to suppress a gcn5 mu-tation. A likely candidate is the RPD3 gene, since mutations inthis gene suppress the Swi5 requirement in the HO gene (35).We therefore measured HO-lacZ activity in single and doublemutants carrying null alleles of the GCN5 and RPD3 genes.FIG. 1. Gcn5 is required for HO expression. (A) Effects of gcn5 disruption on the mRNA levels of the HO, PHO2, and SWI5 genes. Total RNA was extracted fromFY120 (GCN5) and JJY54 (gcn5::hisG) grown in YEPD medium to mid-log phase. ACT1 mRNA was used as a control. (B) Genetic relationships between SWI5 andGCN5. ?-Galactosidase activity was measured in strains carrying an HO-lacZ reporter gene integrated in the chromosome at the HO locus. The strains used were JJY12(wild type [wt]), JJY28 (gcn5::hisG), JJY13 (swi5::hisG), and JJY60 (gcn5::hisG swi5::hisG). Values are averages of three independent measurements with less than 10%deviation.TABLE 1. Yeast strains used in this studyStrainGenotypeFY120...........................MATa ura3-52 leu2?1 his4-912? Lys2-128?RT238...........................MAT? ura3-52 leu2?1 his3 trp1 HO-lacZJJY12............................MAT? ura3-52 leu2 ?1 trp1 lys2-128? HO-lacZJJY13............................Same as JJY12, plus swi5::hisGJJY28............................Same as JJY12, plus gcn5::hisGJJY36............................Same as JJY12, plus sin1?::TRP1JJY42............................Same as JJY28, plus hhf2-8JJY43............................Same as JJY28, plus hhf2-13JJY44............................Same as JJY28, plus sin2-1JJY45............................Same as JJY28, plus sin1?::TRP1JJY54............................Same as FY120, plus gcn5::hisGJJY60............................Same as JJY28, plus swi5::hisGJJY64............................Same as JJY12, plus rpd3?::LEU2JJY65............................Same as JJY28, plus rpd3?::LEU2JJY72............................Same as JJY41, plus rpd3?::LEU2JJY73............................Same as JJY42, plus rpd3?::LEU2JJY74............................Same as JJY43, plus rpd3?::LEU2JJY75............................Same as JJY44, plus rpd3?::LEU21050 PE´REZ-MARTI´N AND JOHNSONMOL. CELL. BIOL.

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The results (Fig. 2) show that a deletion of the deacetylasegene RPD3 alleviates the requirement for the histone acetyl-transferase gene GCN5 in HO gene expression. The level ofsuppression of a gcn5 mutation by the deletion of RPD3 issimilar to that observed in the case of swi5 mutations (35) andis also similar to the suppression observed in a triple swi5 gcn5rpd3 mutant, again supporting the view that SWI5 and GCN5function in the same pathway.One of the defects originally observed in gcn5 mutant strainswas their inability to grow in media imposing amino acid lim-itation (20). Thus, a strain carrying a deletion of the GCN5gene is defective in growth in media containing 3-AT, a con-dition that mimics histidine starvation (5). To address whethera deletion in the RPD3 gene suppresses other defects in gcn5strains, we also tested the ability of the RPD3 deletion to allowgrowth of a gcn5 strain in the presence of 3-AT. As shown inFig. 2, the gcn5 strain exhibited a growth defect under suchconditions compared with an isogenic wild-type strain. Dele-tion of the RPD3 gene indeed alleviates this defect, allowinggrowth of the gcn5 strain under these conditions.Disruption of SIN1, a gene encoding an HMG1-like protein,also suppresses gcn5 defects. In addition to sin3 and rpd3mutations, defects in other genes are well-known suppressorsof transcriptional deficiencies in HO. One of these genes isSIN1. This gene encodes a protein with similarities to themammalian HMG1 protein, and it is believed to be a compo-nent of chromatin (11). We have monitored both HO-lacZexpression and the ability to grow in the presence of 3-AT of adouble mutant defective in both GCN5 and SIN1. The resultsshown in Fig. 3 indicate that the absence of Sin1 protein re-lieves the requirement of Gcn5 both for HO expression and forgrowth on 3-AT.Histone mutations also suppress gcn5 defects. An explana-tion for the results obtained with the sin1 mutant is that thesuppression we observed is caused by a defect in chromatinstructure, such that this defective chromatin bypasses the re-quirement for histone acetylation. If this is the case, then othermutations which produce defective chromatin might also beexpected to suppress the gcn5 defects. Certain amino acidchanges (sin mutations) in either histone H3 or histone H4alleviate the same set of transcriptional defects as does the sin1mutation (12, 23). These sin mutations lie in the histone folddomain of histones H3 and H4, and they are in close proximityto one another on the surface of the histone octamer. It hasbeen proposed that residues altered by these mutations maydefine a functional domain (the SIN domain) that behavesformally as a negative regulator of transcription (12).To address if defective histones also suppress gcn5 muta-tions, the following histone mutant alleles were tested for theirability to suppress a deletion of the GCN5 gene: sin2-1 (R116Hin HHT1), hhf2-7 (R45C in HHF4), hhf2-8 (V43I in HHF4),and hhf2-13 (R45H in HHF4). In spite of the fact that thetargets for GCN5 protein are the histone tails, mutations in thehistone fold are able to efficiently suppress the defects causedby the absence of the GCN5 gene product (Fig. 4A).We also determined the effects of combining a deletion ofthe RPD3 gene with the histone sin mutations. Levels of HO-lacZ activity were determined in single and double mutants,and we found in the double mutants a strong synergistic effect;that is, the activity displayed by the double mutant is higherthan the sum of the activities displayed by the single mutants(Fig. 4B). The same synergistic effect is also seen in combina-tions of rpd3 and sin1 mutations (data not shown).FIG. 2. A deletion of the RPD3 gene partially suppresses the defects caused by a disruption of the GCN5 gene. Cultures of JJY1 (wild type [wt]), JJY64(rpd3?::LEU2), JJY28 (gcn5::hisG), and JJY65 (rpd3?::LEU2 gcn5::hisG) cells (approximately 5 ? 106/ml) were spotted in 10-fold serial dilutions on medium lackinghistidine (SD-HIS) and on medium lacking histidine and containing 10 mM 3-AT. Plates were incubated at 30°C for 3 days. The same cultures were used to measure?-galactosidase activity (in Miller units). Values are averages of three independent measurements with less than 10% deviation.FIG. 3. Deletion of the SIN1 gene alleviates the defects associated with disruption of the GCN5 gene. Cultures of JJY12 (wild type [wt]), JJY36 (sin1?::TRP1),JJY28 (gcn5::hisG), and JJY45 (sin1?::TRP1 gcn5::hisG) cells (approximately 5 ? 106/ml) were spotted in 10-fold serial dilutions on medium lacking histidine (SD-HIS)and on medium lacking histidine and containing 10 mM 3-AT. Plates were incubated at 30°C for 3 days. The same cultures were used to measure ?-galactosidase activity(in Miller units). Values are averages of three independent measurements with less than 10% deviation.VOL. 18, 1998Gcn5 AND CHROMATIN1051

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DISCUSSIONRegulation of the yeast HO gene is complex, and many genesthat regulate HO have been identified (16). These includegenes encoding the SWI/SNF complex (22, 32); SIN1, whichencodes an HMG1-like protein (11); SIN2, which encodeshistone H3; HHF4, which encodes histone H4 (12); and SIN3,which, along with RPD3, is involved in the deacetylation ofhistones (35). In this paper, we show a requirement for theGCN5 gene, which encodes a histone acetyltransferase (3), foroptimal transcription of the HO gene.The identification of histone acetyltransferases and histonedeacetylases as transcriptional regulators provides molecularFIG. 4. Histone sin mutations suppress gcn5 defects. (A) Cultures of JJY12 (wild type [wt]), JJY28 (gcn5::hisG), JJY41 (hhf2-7 gcn5::hisG), JJY42 (hhf2-8gcn5::hisG), JJY43 (hhf2-13 gcn5::hisG), and JJY44 (sin2-1 gcn5::hisG) cells (approximately 5 ? 106/ml) were spotted in 10-fold serial dilutions on medium lackinghistidine (SD-HIS) and on medium lacking histidine and containing 10 mM 3-AT. Plates were incubated at 30°C for 3 days. The same cultures were used to measure?-galactosidase activity (in Miller units). Values are averages of three independent measurements with less than 10% deviation. (B) Effects of rpd3 deletion on thesuppression of gcn5 defects by histone sin mutations and sin1 mutations. The strains used were JJY12, JJY28, and JJY41 through JJY44 (all as described for panel A),as well as JJY65 (rpd3?::LEU2 gcn5::hisG), JJY72 (hhf2-7 rpd3?::LEU2 gcn5::hisG), JJY73 (hhf2-8 rpd3?::LEU2 gcn5::hisG), JJY74 (hhf2-13 rpd3?::LEU2 gcn5::hisG),and JJY75 (sin2-1 rpd3?::LEU2 gcn5::hisG). Values are averages of three independent measurements with less than 10% deviation.1052PE´REZ-MARTI´N AND JOHNSONMOL. CELL. BIOL.